The engineering of bacteria to controllably deliver therapeutics is an attractive application for synthetic biology. While most synthetic gene networks have been explored within microbes, there is a need for further characterization of in vivo circuit behavior in the context of applications where the host microbes are actively being investigated for efficacy and safety, such as tumor drug delivery. One major hurdle in is that culture-based selective pressures are absent in vivo, leading to strain-dependent instability of plasmid-based networks over time. Here, we experimentally characterize the dynamics of in vivo plasmid instability using attenuated strains of S. typhimurium and real-time monitoring of luminescent reporters. Computational modeling described the effects of growth rate and dosage on live-imaging signals generated by internal bacterial populations. This understanding will allow us to harness the transient nature of plasmid-based networks to create tunable temporal release profiles that reduce dosage requirements and increase the safety of bacterial therapies.
Over the past century, the ability of bacteria to accumulate preferentially in tumors has prompted the investigation of the use of a number of strains for cancer therapy, including C. novyi, E. coli, V. cholorae, B. longum, and S. typhimurium ([3], [12], [2], [24], [15], [21]). Attenuated strains of S. typhimurium have generated particular interest as they can innately home in on tumors and colonize a variety of sizes, and have exhibited safety and tolerance in human clinical trials ([22], [5], [19], [8]]). S. typhimurium were initially shown to have anti-tumor effects through recruitment of the host immune system and competition with cancer cells for nutrients. Subsequently, engineered production of therapeutic cargo was added through simple genetic modifications. While these studies represent important advances in the use of bacteria for tumor therapies, the majority have relied on constitutive, “always on” cargo production ([9], [23], [14], [6]) that typically results in high dosages, off-target effects, and development of host resistance.
As a next step, synthetic biology seeks to add controlled and dynamic production of cargo by utilizing computationally-designed “circuits” that have sophisticated sensing and delivery capability ([7], [4], [17]s[1]). These circuits can be designed to act as delivery systems that sense tumor-specific stimuli and self-regulate cargo production as accessory. Since plasmids are the common framework for synthetic circuits, we begin by characterizing the dynamics of plasmid-based gene expression in vivo by utilizing real-time luminescence imaging, quantitative biodistribution measurement, and computational modeling. Together, these approaches provide a framework for exploiting the inherent instability of plasmid-based networks, which will facilitate the generation of specific temporal release profiles directly within the tumor environment.